ABSTRACT Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), complete a tightly controlled sequence of events during differentiation to recapitulate functional cells from our bodies. This in vitro maturation process initiates by induction of mesoderm, ectoderm, and endoderm lineage progenitors, and provides unique opportunities for disease modeling, drug screening, cell- based therapies, and uncovering mechanisms in cell development. Yet, key obstacles remain and include (1) inefficient PSC differentiation, (2) costly, labor-intensive protocols that promote genetic aberrations, and (3) developmental immaturity and reduced functionality of terminal cells. Mechanistically, failed erasure of epigenetic memory leaves residual marks that affects chromatin structure and gene expression from a hiPSC's somatic cell type of origin. Thus, it is imperative to understand and implement optimized in vitro hPSC differentiation protocols that reconstitute gene regulation and epigenome configuration programs matching in vivo cell counterparts to achieve superior cell types for all applications that employ this remarkable system. The role of cytokines, growth factors, and chemokines in driving hPSC differentiation is firmly established in cell fate determination. Additionally, the facilitating role of metabolic flux and metabolite levels in reconfiguring the epigenome to improve hPSC differentiation has recently been uncovered. Yet, a factor generated by metabolic activity?acidification?has not been examined as a microenvironment stimulus controlling hPSC differentiation despite its known roles in driving de- and re-differentiation during pathogenic and physiological development. We recently discovered both differential metabolic programs and extracellular acidification rates during ectoderm and mesoderm lineage differentiation. This confirms a direct connection between metabolism and acidification, and further suggests a role for lowered pH in early lineage fate acquisition. The overall study goal here is to uncover pH-dependent mechanisms in lineage-specific cell fate, and to ultimately exploit these processes for optimized hPSC differentiation. To test the hypothesis that pH-sensitive mechanisms control early cell fate, we will embark on 3 specific aims: (1) To determine the importance of pH in lineage partitioning under spontaneous PSC differentiation in an embryoid body model. (2) To examine remodeling of epigenome configuration and resulting lineage-specific gene expression to identify pH-sensitive regulators governing early differentiation under low pH using transcriptomic and epigenetic screens. And (3) to study the feasibility of pH modulation to enrich for functional mesoderm derivatives compared to current hPSC differentiation methodologies. Success in these studies will open new pathways through pH manipulations for generating superior in vitro models of early human development for numerous promising applications in health and disease.